The MerR-Like Regulator BrlR Confers Biofilm Tolerance by Activating Multidrug Efflux Pumps in Pseudomonas aeruginosa Biofilms

A defining characteristic of biofilms is antibiotic tolerance that can be up to 1,000-fold greater than that of planktonic cells. InPseudomonas aeruginosa , biofilm tolerance to antimicrobial agents requires the biofilm-specific MerR-type transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm tolerance has not been elucidated. Genome-wide transcriptional profiling indicated thatbrlR was required for maximal expression of genes associated with antibiotic resistance, in particular those encoding the multidrug efflux pumps MexAB-OprM and MexEF-OprN. Chromatin immunoprecipitation (ChIP) analysis revealed a direct regulation of these genes by BrlR, with DNA binding assays confirming BrlR binding to the promoter regions of themexAB-oprM andmexEF-oprN operons. Quantitative reverse transcriptase PCR (qRT-PCR) analysis further indicated BrlR to be an activator ofmexAB-oprM andmexEF-oprN gene expression. Moreover, immunoblot analysis confirmed increased MexA abundance in cells overexpressingbrlR . Inactivation of both efflux pumps rendered biofilms significantly more susceptible to five different classes of antibiotics by affecting MIC but not the recalcitrance of biofilms to killing by bactericidal agents. Overexpression of either efflux pump in a ΔbrlR strain partly restored tolerance of ΔbrlR biofilms to antibiotics. Expression ofbrlR in mutant biofilms lacking both efflux pumps partly restored antimicrobial tolerance of biofilms to wild-type levels. Our results indicate that BrlR acts as an activator of multidrug efflux pumps to confer tolerance toP. aeruginosa biofilms and to resist the action of antimicrobial agents.